Blood can be fractionated into several component parts. One of these component parts, platelet rich plasma (PRP) is blood plasma that has been enriched for platelets. As a concentrated source of autologous platelets, PRP contains (and releases through degranulation) several different growth factors and other cytokines that stimulate healing of bone and soft tissue. The efficacy of certain growth factors in healing various injuries and the concentrations of these growth factors found within PRP are the theoretical basis for the use of PRP in tissue and bone repair. The platelets collected in PRP can be activated by the addition of thrombin and calcium chloride, which induces the release of these factors from alpha granules. PRP and other blood extracts also contain other proteins, such as collagen, fibrinogens and fibronectin, amongst others, that have both signaling and scaffolding properties. Upon delivery of PRP to a tissue or bone defect, these proteins can form a gel that serves as a provisional matrix (e.g., a scaffold) that can support tissue or bone regeneration. Thus, PRP and other blood extracts can support two sometimes overlapping functions: delivery of growth factors and establishment of a scaffold to support tissue or bone regeneration.
There are, at present, two methods of PRP preparation approved by the U.S. Food and Drug Administration. Both processes involve the collection of whole blood that is anticoagulated with citrate dextrose before undergoing two stages of centrifugation designed to separate the PRP aliquot from platelet-poor plasma and red blood cells. In humans, the typical baseline blood platelet count is approximately 200,000 per μL. Therapeutic PRP concentrates the platelets by roughly five-fold. There is however broad variability in the production of PRP by various concentrating equipment and techniques.
In humans, PRP has been investigated and used as a clinical tool for several types of medical treatments, including pain, sports injury, nerve injury, tendinitis, osteoarthritis, cardiac muscle injury, bone repair and regeneration, plastic surgery, and oral surgery.
In clinical settings, PRP is delivered to the body in one of two ways, depending on the specific application. For open wounds (surgical procedures, diabetic ulcers, etc.), PRP is activated by the addition of thrombin and calcium chloride, which results in the release of clotting factors that cause the PRP to coagulate into a gelatinous form. The activation of PRP in these settings both stimulates the release of growth factors and allows for the material to be held in place during clinical procedures. For applications that involve PRP injections into tissues (joints injuries, tendonitis, arthritis, etc.), pre-activation of the PRP with thrombin and calcium chloride is neither required nor advantageous. Platelets are naturally activated when they come in contact with extracellular matrix proteins like collagen, so activation of the PRP occurs spontaneously when injected directly into tissues. Maintaining the PRP in an inactivated (uncoagulated) state makes syringe-based injections possible. Moreover, spontaneous activation of the PRP upon contact with the tissue results in localized growth factor release.
Although PRP has shown great promise to improve medical outcomes for a wide range of clinical applications, alternative delivery mechanisms of the PRP could greatly improve its handling properties as well as enhance its medicinal qualities. Medical field of PRP delivery relies entirely on the two techniques described above: direct injection into tissues or induced PRP activation/coagulation.
Current PRP technology has several limitations, including: the handling properties of PRP gels cannot be controlled; the physical properties of a gel formed by activation of PRP cannot be controlled; degradation rates of PRP gels cannot be controlled; release rates of biologically active components of the PRP cannot be controlled; the water content of PRP gels cannot be controlled; and immediate activation of platelets is required.
In addition to the activation of platelets by thrombin and calcium chloride, other methods are available that can also cause the release of growth factors from platelets, including the activation of granulation using PAR peptides or ADP, and lysis or disruption of platelets through chemical or mechanical means. Further, blood derived growth factors may be obtained from whole blood or other blood fractions such as serum or plasma and may also be further purified by standard biochemical methods. However, in many cases, these methods do not lead to clot formation, and thus do not provide the gelatinous material that would otherwise provide a matrix for the growth factors or a scaffold to support tissue or bone regeneration.
Furthermore, the provisional matrix (or scaffold) provided by a blood extract that would support tissue or bone regeneration may have inconsistent mechanical and/or biological properties, such as in the case of PRP, or may be incapable of formation in the case of other extracts.
For these reasons, synthetic matrices have been developed to administer blood extracts. For example, attempts have been made to deliver PRP, platelets, and platelet extracts in gelatin and alginate hydrogels, PLA/PRP gel composite scaffolds, porous PLGA/PRP gel composite scaffolds, and electrospun PRP/polymeric fiber composites. In other examples, whole blood is mixed with chitosan/glycerin phosphate or a blood extract is mixed with a peg-diacrylate scaffold. Sell, S. et al. “The Incorporation and Controlled Release of Platlet-Rich Plasma-Derived Biomolecules from Polymeric Tissue Engineering Scaffolds,” Polym. Int. 61:1703-1709. Unfortunately, each of these methods suffers from one or more of the following limitations:
1. Inconsistent Mechanical Properties Resulting from a Reliance of the Fibrin Clot to Contribute to the Structure of the Scaffold.
As mentioned above, activation of PRP using, for example, thrombin will lead to the formation of a gelatinous gel (a clot). In traditional methods of PRP delivery, this clot serves as the scaffold for tissue or bone regeneration. However, this scaffold will have variable properties due to factors such a patient-to-patient variations and inconsistent processing techniques. To address these limitations, prior methods have augmented the clot with synthetic polymers such as PLA or PLGA. However these methods still rely on the clot to serve as a substantial component of the scaffold, which results in an improved, but still inconsistent and variable, scaffold. A fully synthetic polymer that would provide a consistent scaffold that is not reliant on the properties of the clot would be advantageous.
2. Technically Difficult Processing Techniques that May Prevent Near-Patient Processing and Application.
Some existing methods require techniques such as electrospinning of a platelet extract prior to use in an individual. These methods are time consuming, require specialized equipment, and are generally inconsistent with near-patient processing applications. A method that is rapidly performed at the site of the intended use would be beneficial.
3. Requirement For Specialized Equipment and Trained Personnel.
Even relatively simple techniques, such as the use of PRP, require specialized equipment, e.g., to separate the PRP from other components in the blood. Further, a trained transfusionist is often required to draw the blood and perform the separation. It would be desirable to have a method that uses only standard and readily available equipment and personnel that are already in place in most doctor's offices and clinics.
4. Growth Factor Release Properties that are not Responsive to the Temporal and Spatial Demands of the Tissue or Bone Regeneration Processes.
A problem with most existing methods is that upon application to an individual, the growth factors that promote tissue or bone regeneration will diffuse away from the application site (e.g., the wound) in a manner that is independent of the rate of healing. Thus, although growth factors with certain existing methods are present at the intended site at the time of initial application, they are not maintained within the desired site of action throughout the healing process, which limits the overall effectiveness of prior methods. It would be desirable to have a scaffold that retains growth factors at the desired site of action (e.g., at the wound) and releases the growth factors in response to new tissue formation.
5. Scaffold Degradation Rate not Responsive to the Tissue Regeneration Processes.
Similarly, the scaffold of certain existing methods will degrade in a manner that is independent of the rate of tissue formation. Premature degradation does not permit the injury to benefit from the scaffold throughout the healing process. It would be desirable to utilize a scaffold that degrades as new tissue is formed.
6. Inconsistent Degradation Products that May be Inefficiently Cleared.
Because certain prior methods do not have a regular, clearly defined structure, the degradation products that result from these methods are often are poorly defined and have a large size distribution. The large size distribution can present a particular problem as large degradation products may be inefficiently cleared and place an additional burden on an individual's system. A scaffold that leads to small, well defined degradation products that can be efficiently cleared would be beneficial.
7. Product does not Conform to Tissue (or Bone) Defect Resulting in Poor Integration with Native Tissue at Wound Margins.
Many of the existing products are formed outside of the tissue defect and are then cut or otherwise manipulated in an attempt to conform to the shape of the tissue defect. However, it is impossible to perform such shaping precisely enough that it fully conforms to the contours of the defect (e.g., to the shape of a wound). The resulting gaps then become an impediment to the movement into the scaffold of cells, proteins and other factors needed to facilitate tissue or bone regeneration. Moreover, gaps present an increased opportunity for bacteria or other undesirable materials to enter the defect site and lead to complications or otherwise slow or impede healing. A material that is formed in situ and more fully conforms to the shape of the tissue or bone defect (e.g., by forming a contiguous boundary with the edges of the defect) would be desirable.
8. Poor Control of Polymerization (Gelation) Process During Administration.
Certain existing methods are allowed to form a scaffold within the defect. However, these methods rely on the formation of a gelatinous clot. As previously noted, the time for clotting can be highly variable and can also lead to inconsistent results. For example, in many cases, the clotting process will have started before the material is fully administered. In other cases, the material may polymerize too slowly. A material that can be placed in the defect and then rapidly polymerized in situ using a process that provides precise temporal and spatial control would be highly beneficial.
9. Altered Activity of the Growth Factors and Other Proteins that May be Important to Tissue or Bone Regeneration.
Finally, many of the existing methods require steps that may alter the activity of growth factors in a blood extract. For example, processes that disrupt platelets through a freeze-thaw cycle or denaturation steps can substantially alter growth factor activity and lead to decreased efficacy when used in a method of tissue or bone regeneration. Similarly, methods such as electrospray fabrication can substantially alter the activity of the growth factors. A method that does not require the use of conditions that can alter the activity of growth factors or other proteins in a blood extract would be beneficial. There is thus a continued need in the art for compositions that address the limitations of current PRP and blood extract administration techniques.